The disclosed invention is in the general field of oligonucleotide synthesis and purification.
Oligonucleotides are being pursued as therapeutic and diagnostic agents. Examples of such oligonucleotides include antisense oligonucleotides, aptamers, triplex forming agents, external guide sequences, catalytic oligomers, and ribozymes. Synthetic oligonucleotides based on oligo(2′-O-allylribonucleotide)s which can cleave, or induce cleavage of, specific RNA molecules are showing great promise as a new class of rationally designed therapeutics. Such chemically modified oligonucleotides are useful for cleaving an unwanted or overexpressed RNA in a highly specific fashion, thus preventing the synthesis of the corresponding protein which is causative for a particular disease state. Such an oligonucleotide having RNA cleaving activity has been successfully used in mice (Lyngstadaas et al., EMBO Journal 14:5224-5229 (1995)). Several other examples of such compounds have been shown to possess reasonable pharmacokinetics (Desjardins et al., J. Pharmacol. Exp. Ther. 278:1419-1427 (1966)). Versions of these compounds that are stable enough for in vivo applications, yet have reasonable catalytic activity, contain five residual purine ribonucleotides at critical positions in the hammerhead catalytic core and are generally in the range of 34 to 38 residues in length (Lyngstadaas et al., EMBO Journal 14:5224-5229 (1995)).
Synthetic methods for preparing oligonucleotides generally include solid phase synthesis using phosphoramidite coupling chemistry. This chemistry generally involves coupling the 3′-hydroxy group of a first nucleotide to a solid support, and reacting the 5′-hydroxy group with subsequent monomers containing protected 5′-hydroxy groups. After each coupling step, the 5′-protecting group is removed, freeing up a subsequent 5′-hydroxy group for subsequent coupling with an additional monomer. The coupling chemistry has a relatively high yield for each step, but about one to two percent of the hydroxy groups are not successfully coupled at each step in the synthesis. These un-reacted hydroxy groups must be prevented from further reactions, to avoid the preparation of unwanted sequences. To accomplish this, the hydroxy groups are capped, for example, using acetic anhydride. The capped sequences are typically referred to as failure sequences.
A major synthetic hurdle is the separation of failure sequences from the desired oligonucleotide. Current purification methodologies include anion-exchange HPLC (high pressure liquid chromatography), column chromatography, polyacrylamide gel electrophoresis, reversed phase chromatography, ion-pair chromatography, and affinity chromatography.
Purification of oligoribonucleotides and/or chemically modified ribozymes using anion-exchange chromatography with either Pharmacia Mono Q or Dionex NucleoPac columns and eluting with a salt gradient such as either sodium or lithium perchlorate has been described (Sproat et al., Nucleosides & Nucleotides 14:255-273 (1995); Wincott et al., Nucleic Acids Research 23:2677-2684 (1995)). Single base resolution of oligonucleotides, such as those described above, is typically not possible using these methods.
Additional problems associated with this purification technique are problematic secondary structure for longer RNA molecules (thus, purifications must be done at higher temperature to denature the RNA), low loading capacity, organic co-solvents such as acetonitrile are often necessary, and the columns are very expensive. Thus, the use of current ion-exchange HPLC methods is problematic even for purifying oligonucleotides on the scale of a few milligrams.
A popular method for purifying chemically synthesized oligodeoxyribonucleotides has been to leave the 5′-O-dimethoxytrityl (DMTr) protecting group on following the last coupling step of the synthesis. This protecting group functions as a lipophilic handle. The handle allows purification of full length material away from non-full length material using reversed phase HPLC because the non-full length molecules do not contain the protecting group and thus have a much less lipophilic character than the full length molecules. Unfortunately, when additional lipophilic chemical modifications are present in the oligonucleotide, or if the oligonucleotide is long (effectively increasing the lipophilicity of the non-full length molecules), undesired molecules may be more strongly retained and may even merge with the product peak, thus rendering the purification difficult or impossible. Attempts have been made to overcome this limitation by making the trityl group more lipophilic, either by adding a long alkyl or alkoxy substituent (Seliger et al., Tetrahedron Letters 2115-2118 (1978); Gortz and Seliger, Angew. Chem., Int. Ed. Engi. 20:681 (1981); Seliger and Schmidt, J. Chromatogr. 397:141-151 (1987); Gupta et al., J. Chromatogr. 541:341-348 (1991)), by adding butyl groups to all three aromatic rings of the trityl moiety, or by adding additional aromatic rings (Ramage and Wahl, Tetrahedron Letters 34:7133-7136 (1993)). In the latter case the 4-(17-tetrabenzo[a,c,g,i]fluorenylmethyl)-4′,4″-dimethoxytrityl group was employed to aid in the purification of long oligodeoxyribonucleotides greater than 100 residues in length. However, a disadvantage to this approach is that the modified trityl groups are significantly more acid labile than the standard DMTr group.
Purification of chemically synthesized oligoribonucleotides and 2′-O-methyl modified hammerhead ribozymes has been achieved by the trityl-on method followed by a second purification step by ion-exchange HPLC (Wincott et al., Nucleic Acids Research 23:2677-2684 (1995)). However, this method is very time consuming and due to the intrinsic instability of the trityl group losses occur due to premature detritylation. Furthermore, in any purification strategy the potential for product loss is increased with each successive manipulation of the product. In order for the majority of the lipophilicity of the oligonucleotide to be derived from the trityl group (which is necessary for efficient trityl based purification), the other lipophilic groups on the synthetic RNA (such as the silyl protecting groups) must be removed (Wincott et al., Nucleic Acids Research 23:2677-2684 (1995)). Unfortunately, the preferred desilylating agents, such as triethylamine trihydrofluoride, also cause loss of the DMTr group. However, this problem can be solved using triethylamine trihydrofluoride/triethylamine/N-methylpyrrolidinone which is not acidic enough to cleave the DMTr groups (Wincott et al., Nucleic Acids Research 23:2677-2684 (1995)).
Another problem associated with this method is that the oligonucleotide must be precipitated before the HPLC purification to avoid destroying the HPLC column silica matrix with the fluoride reagent used to remove the silyl groups. Also, following purification, the trityl group must be removed under acidic conditions. Care must be taken to avoid possible acid catalyzed migration of the 3′,5′-internucleotide linkages at ribose positions as well as strand cleavage (Mullah and Andrus, Nucleosides & Nucleotides 15:419-430 (1996)). For these reasons, the above HPLC method is not entirely satisfactory for obtaining high yields of very pure oligonucleotides.
Reverse-phase ion-pair chromatography, whereby separations are based on both hydrophobicity and on the number of anions associated with the molecule, has been used to purify fully deprotected oligoribonucleotides in a single HPLC step. However, capacity and resolution are relatively low on silica based matrices (Murray et al., Analytical Biochemistry 218:177-184 (1994)). New non-porous, inert polymer resins have been used to address the problem of low loading and have provided reasonable separations of oligodeoxyribonucleotides with loadings of 1 mg/ml of resin. Nonetheless, run times on the order of 2 hours are required to give adequate resolution (Green et al., BioTechniques 19:836-841 (1995)). Likewise, reverse-phase ion-pair chromatography on polystyrene based columns such as the PRP-1 column from Hamilton has been used to purify chemically synthesized unprotected chimeric hammerhead ribozymes (Swiderski et al., Analytical Biochemistry 216:83-88 (1994)). This method takes advantage of the tetrabutylammonium ions present after the desilylation reaction. Although the columns have a reasonable capacity, resolution between full length product and failure sequences differing in length by one or two residues is poor for oligonucleotides containing 38 or more residues.
Polyacrylamide gel electrophoresis can be used for the small scale purification of chemically modified ribozymes. A method for larger scale purification which combines gel electrophoresis with column chromatography has been recently described (Cunningham et al., Nucleic Acids Research 24:3647-3648 (1996)) and was used to purify 6.5 mg of a 34 residue ribozyme in a single run. However, the method is limited because it requires about 20 hours to complete, is limited to relatively small amounts of material, and uses relatively toxic chemicals to prepare the gels.
An alternative purification strategy is based on affinity methods, whereby a ligand-receptor interaction is utilized such as the biotin-streptavidin pair. A 5′-biotinylated oligonucleotide can be captured on an agarose column bearing covalently attached streptavidin. Assuming that the linkage between the biotin moiety and the oligonucleotide is cleavable, such as a disulfide linkage, the desired oligonucleotide can be eluted from the column by washing the column with a reagent that cleaves the linkage. In the case of a disulfide linkage, a reducing agent such as dithiothreitol or β-mercaptoethanol would be used. An alternative method using a photocleavable biotin containing moiety has also been described (Olejnik et al., Nucleic Acids Research 24:361-366 (1996)). Alternative oligodeoxyribonucleotides carrying a 5′-biotinylated trityl group have been affinity captured on streptavidin agarose, where the fully deprotected pure oligonucleotide is released by acid treatment (Gildea et al., Tetrahedron Letters 31:7095-7098 (1990)). These methods are adequate for small scale purification, but the expense of the streptavidin agarose limits their application to small scale purifications.
Another form of affinity purification uses immobilized sequences complementary to the 5′- or 3′-end of the material to be purified. Such a method has been used for the batchwise purification of specific tRNAs (Tsurui et al., Analytical Biochemistry 221:166-172 (1994)). A limitation of this method is that it requires a different affinity matrix for every oligonucleotide synthesized.
It is therefore an object of the present invention to provide methods and compositions for purifying oligomeric molecules, such as oligonucleotides, without the problems associated with long purification times, acid labile protecting groups, and overlap on the column between failure sequences and the desired product peak.